Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter.

Conformational changes in the human Na(+)/glucose cotransporter (hSGLT1) were examined using hSGLT1 Q457C expressed in Xenopus laevis oocytes and tagged with tetramethylrhodamine-6-maleimide (TMR6M). Na(+)/glucose cotransport is abolished in the TMR6M-labeled mutant, but the protein binds Na(+) and sugar [Loo et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 7789-7794]. Under voltage clamp the fluorescence of labeled Q457C was dependent on external cations. Increasing [Na(+)] increased fluorescence with a Hill coefficient of 2 and half-maximal concentration (K(Na)(0.5)) of 49 mM at -90 mV. Li(+) also increased fluorescence, whereas choline, tetraethylammonium, and N-methyl-D-glucamine did not. Fluorescence was increased by sugars with specificity: methyl alpha-D-glucopyranoside > D-glucose > D-galactose > D-mannitol. Voltage-jump experiments (in 100 mM NaCl buffer in absence of sugar) elicited parallel changes in pre-steady-state charge movement and fluorescence. Charge vs voltage and fluorescence vs voltage curves followed Boltzmann relations with the same median voltage (V(0.5) = -50 mV), but the apparent valence was 1 for charge movement and 0.4 for fluorescence. V(0.5) for fluorescence and charge movement was shifted by -100 mV per 10-fold decrease in [Na(+)]. Under Na(+)-free conditions, there was a voltage-dependent change in fluorescence. Voltage-jump experiments showed that the maximal change in fluorescence increased 20% with sugar. These results indicate that Na(+), sugar, and membrane voltage change the local environment of the fluorophore at Q457C. Our interpretation of these results is (1) the conformational change of the empty transporter is voltage dependent, (2) two Na(+) ions can bind cooperatively to the protein before sugar, and (3) sugar binding induces a conformational change.     

Conformational dynamics of hSGLT1 during Na+/glucose cotransport

This study examines the conformations of the Na(+)/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na(+) buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants tau(med) (3-20 ms, medium) and tau(slow) (15-70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (DeltaF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (DeltaF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zdelta (apparent valence of voltage sensor) approximately 1; and V(0.5) (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na(+)/glucose cotransport), and reduced maximal charge (Q(max)) and fluorescence (DeltaF(max)) with half-maximal concentrations (K(0.5)) of 1 mM. Increasing [alphaMDG](o) also shifted the V(0.5) for Q and DeltaF to more positive values, with K(0.5)'s approximately 1 mM. The major difference between Q and DeltaF was that at saturating [alphaMDG](o), the presteady-state current (and Q(max)) was totally abolished, whereas DeltaF(max) was only reduced 50%. Phlorizin reduced both Q(max) and DeltaF(max) (K(i) approximately 0.4 microM), with no changes in V(0.5)'s or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na(+)/sugar cotransport (saturating voltage and external Na(+) and sugar concentrations) is the slow release of Na(+) from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.     

Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter.

The human Na(+)-glucose cotransporter (hSGLT1) has been shown to generate, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials once the voltage had reached a new constant value. By the cut-open oocyte technique (voltage rising-speed approximately 1 mV/microsecond), phlorizin-sensitive transient currents could be detected with a higher time resolution during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+]o), phlorizin-sensitive currents exhibited two relaxation time-constants: tau 1 increased from 2 to 10 ms when Vm decreased from +60 mV to -80 mV and remained at 10 ms for more negative Vm; tau 2 ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a previously proposed model, these two time constants could be accounted for by 1) Na+ crossing a fraction of the membrane electrical field to reach its binding site on the carrier and 2) conformational change of the free carrier. To test this hypothesis, the time constants were measured as [Na+]o was progressively reduced to 0 mM. At 30 and 10 mM external Na+, tau 1 reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0). Under these conditions, tau 1 continuously increased through the whole voltage range and did not reach the 10 ms level even when Vm had attained -200 mV while tau 2 remained in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must occur in more than one step and 2) Na+ binding/debinding is not responsible for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the portion of the hSGLT1 transport cycle involving extracellular Na+ binding/debinding and the dual-step conformational change of the free carrier, tau 1 and tau 2 were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. This study shows that 1) tau 1 and tau 2 correspond to two steps in the conformational change of the free carrier, 2) Na+ binding/debinding modulates the slow time constant (tau 1) and 3) a voltage-independent slow conformational change of the free carrier accounts for the observed plateau value of 10 ms.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.     

Position 170 of Rabbit Na+/glucose cotransporter (rSGLT1) lies in the Na+ pathway; modulation of polarity/charge at this site regulates charge transfer and carrier turnover

Positions 163, 166, and 173, within the putative external loop joining transmembrane segments IV and V of rabbit Na(+)/glucose cotransporter, form part of its Na(+) interaction and voltage-sensing domain. Since a Q170C mutation within this region exhibits anomalous behavior, its function was further investigated. We used Xenopus oocytes coinjected with mouse T-antigen to enhance Q170C expression, and the two-microelectrode voltage-clamp technique. For Q170C, alpha-methyl D-glucopyranoside, phloridzin, and Na(+) affinity values are equivalent to those of wild-type; but turnover is reduced approximately 50%. Decreased [Na(+)] reduces Q170C, but not wild-type, charge transfer. Q170C presteady-state currents exhibit three time constants, tau, identical to wild-type. MTSES decreases maximal alpha-methyl D-glucopyranoside-induced currents by approximately 64% and Na(+) leak by approximately 55%; phloridzin and Na(+) affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by approximately 60-70%. MTSEA and MTSET protect against MTSES, but neither affect Q170C function. MTSES has no obvious effect on the tau-values. Q170A behaves the same as Q170C. The mutation Q170E affects voltage sensitivity and reduces turnover, but also appears to influence Na(+) interaction. We conclude that 1), glutamine 170 lies in the Na(+) pathway in rabbit Na(+)/glucose cotransporter and 2), altered polarity and charge at position 170 affect a cotransporter conformational state and transition, which is rate-limiting, but probably not associated with empty carrier reorientation.     

Investigating the conformational states of the rabbit Na+/glucose cotransporter

The Na(+) and voltage-dependence of transient rabbit Na(+)/glucose cotransporter (rSGLT1) kinetics was studied with the two-electrode voltage-clamp technique and Xenopus laevis oocytes. Using step changes in membrane potential, in the absence of glucose but with 100 or 10 mM Na(+), transient currents were measured corresponding to binding/debinding of Na(+) and conformational changes of the protein. Previously, only a single time constant has been published for rSGLT1. We, however, observed three decay components; a fast (tau(f), 0.5-1 ms) voltage- and Na(+)-independent decay, and medium (tau(m), 0.5-4 ms) and slow (tau(s), 8-50 ms) voltage- and Na(+)-dependent decays. Transient currents were simulated and fit using a four-state model to obtain kinetic parameters for the system. The four-state model was able to reconstitute an assortment of experimental data.     

Time-dependent transients in an ionically based mathematical model of the canine atrial action potential

Ionically based cardiac action potential (AP) models are based on equations with singular Jacobians and display time-dependent AP and ionic changes (transients), which may be due to this mathematical limitation. The present study evaluated transients during long-term simulated activity in a mathematical model of the canine atrial AP. Stimulus current assignment to a specific ionic species contributed to stability. Ionic concentrations were least disturbed with the K(+) stimulus current. All parameters stabilized within 6-7 h. Inward rectifier, Na(+)/Ca(2+) exchanger, L-type Ca(2+), and Na(+)-Cl(-) cotransporter currents made the greatest contributions to stabilization of intracellular [K(+)], [Na(+)], [Ca(2+)], and [Cl(-)], respectively. Time-dependent AP shortening was largely due to the outward shift of Na(+)/Ca(2+) exchange related to intracellular Na(+) (Na) accumulation. AP duration (APD) reached a steady state after approximately 40 min. AP transients also occurred in canine atrial preparations, with the APD decreasing by approximately 10 ms over 35 min, compared with approximately 27 ms in the model. We conclude that model APD and ionic transients stabilize with the appropriate stimulus current assignment and that the mathematical limitation of equation singularity does not preclude meaningful long-term simulations. The model agrees qualitatively with experimental observations, but quantitative discrepancies highlight limitations of long-term model simulations.     

Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s?1) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.     

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na(+)/glucose cotransporter (SGLT1) using Na(+)- and H(+)-selective electrodes and with the electroneutral H(+)/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na(+)/glucose influx, which generated a measurable decrease in the trap Na(+) concentration ([Na(+)](T)) by 0.080 +/- 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na(+)](T), which was proportional to the increased cotransport current. In a Na(+)-free, weakly buffered solution (pH 5.5), H(+) drives glucose transport through SGLT1, and this was monitored with a H(+)-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na(+)](T) or [H(+)](T) were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na(+)](T) or [H(+)](T) is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.     

Kinetics of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier

Sodium current and intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). Sodium current activation followed a single-exponential time course, provided a delay was interposed between the onset of the step ON depolarization and that of the current change. The delay decreased with increased ON depolarization and, for a constant ON depolarization, increased with prehyperpolarization. ON charge movement followed a single-exponential time course with time constants tau Q,ON slightly larger than tau Na, ON. For pulses between -70 and -50 mV, tau Q,ON/tau Na,ON = 1.14 +/- 0.08. The OFF charge movement and OFF sodium current tails after a depolarizing pulse followed single-exponential time courses, with tau Q, OFF larger than tau Na, OFF. tau Q,OFF/tau Na,OFF increased with OFF voltage from 1 near -100 mV to 2 near -160 mV. At a set OFF potential (-120 mV), both tau Q,OFF and tau Na,OFF increased with ON pulse duration. The delay in INa activation and the effect of ON pulse duration on tau Q,OFF and tau Na,OFF are inconsistent with a simple two-state, single-transition model for the gating of batrachotoxin-modified sodium channels.     

GAT1 (GABA:Na+:Cl-) cotransport function. Kinetic studies in giant Xenopus oocyte membrane patches.

To explain cotransport function, the "alternating access" model requires that conformational changes of the empty transporter allow substrates to bind alternatively on opposite membrane sides. To test this principle for the GAT1 (GABA:Na+:Cl-) cotransporter, we have analyzed how its charge-moving partial reactions depend on substrates on both membrane sides in giant Xenopus oocyte membrane patches. (a) "Slow" charge movements, which require extracellular Na+ and probably reflect occlusion of Na+ by GAT1, were defined in three ways with similar results: by application of the high-affinity GAT1 blocker (NO-711), by application of a high concentration (120 mM) of cytoplasmic Cl-, and by removal of extracellular Na+ via pipette perfusion. (b) Three results indicate that cytoplasmic Cl- and extracellular Na+ bind to the transporter in a mutually exclusive fashion: first, cytoplasmic Cl- (5-140 mM) shifts the voltage dependence of the slow charge movement to more negative potentials, specifically by slowing its "forward" rate (i.e., extracellular Na+ occlusion); second, rapid application of cytoplasmic Cl- induces an outward current transient that requires extracellular Na+, consistent with extracellular Na+ being forced out of its binding site; third, fast charge-moving reactions, which can be monitored as a capacitance, are "immobilized" both by cytoplasmic Cl- binding and by extracellular Na+ occlusion (i.e., by the slow charge movement). (c) In the absence of extracellular Na+, three fast (submillisecond) charge movements have been identified, but no slow components. The addition of cytoplasmic Cl- suppresses two components (tau < 1 ms and 13 micros) and enables a faster component (tau < 1 micros). (d) We failed to identify charge movements of fully loaded GAT1 transporters (i.e., with all substrates on both sides). (e) Under zero-trans conditions, inward (forward) GAT1 current shows pronounced pre-steady state transients, while outward (reverse) GAT1 current does not. (f) Turnover rates for reverse GAT1 transport (33 degrees C), calculated from the ratio of steady state current magnitude to total charge movement magnitude, can exceed 60 s(-1) at positive potentials.     

A glutamine to glutamate mutation at position 170 (Q170E) in the rabbit Na+/glucose cotransporter, rSGLT1, enhances binding affinity for Na+

Using cysteine mutagenesis and chemical modification by methanethiosulfonate derivatives, it was demonstrated that the external putative loop, joining transmembrane segments (TM's) IV-V of rabbit Na+/glucose cotransporter, rSGLT1, forms part of a Na+ binding and voltage sensing domain. Within this region, exposure to cationic (2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) inhibited F163C, A166C, and L173C, but anionic sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) had no effect. Unexpectedly, MTSEA had no effect on Q170C; however, MTSES profoundly altered Q170C charge transfer and turnover, leaving Na+ and sugar binding affinity unchanged, but mutation of glutamine to anionic glutamate (Q170E) shifted V(0.5) to positive potentials, suggesting enhanced Na+ affinity. To clarify the role of glutamine 170 in Na+ interaction, we embarked on a more detailed investigation of Q170E using the two-microelectrode voltage clamping in Xenopus oocytes. Compared to wild-type (wt) rSGLT1, Q170E exhibits (i) a 2-fold decrease in methyl alpha-D-glucopyranoside affinity (-150 to -90 mV), (ii) a 5-fold increase in Na+ affinity (-150 to -100 mV) with less voltage dependency, (iii) reduced Na+ leak, and (iv) two transient current decay constants (tau(fast), tau(slow)) compared to three (tau(fast), tau(medium), tau(slow)) for wt, and computer simulation of Q170E pre-steady-state currents with a four-state kinetic model yields parameters similar to wt SGLT1, except for a reduced Na+ debinding rate constant compared to wt. Taken together, the data strengthen the conclusion that residue 170 lies in the Na+ pathway and provide the first evidence that it participates in determining Na+ binding.     

Extracellular pH modulates kinetics of the Na(+),K(+)-ATPase

To investigate effects of pH on the Na(+),K(+)-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K(+), and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na(+), K(+)-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na(+) ions with the pump molecule and/or conformational changes during Na(+)/Na(+) exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K(+) (< or =2 mM) and with 50 Na(+). This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na(+). External stimulation of the pump by K(+) in the absence of Na(+) can be described by a voltage-dependent K(m) value with an apparent valency z(K). At higher external pH the z(K) value is reduced. The transient current signal in the absence of external K(+) can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 micros and less than 100 micros during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about -120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K(+) and Na(+) binding.     

Functional asymmetry of the human Na+/glucose transporter (hSGLT1) in bacterial membrane vesicles

The functional characteristics of the forward and reverse transport modes of the human Na(+)/glucose transporter (hSGLT1) were investigated using plasma membrane vesicles of E. coli expressing the recombinant transporter. Correctly and inverse-oriented vesicles were employed to measure the initial rates of methyl-alpha-D-glucose uptake, under zero-trans conditions, as a function of Na(+), sugar, and phlorizin concentrations and membrane potential. This approach enabled the analysis of the two faces of hSGLT1 in parallel, revealing the reversibility of Na(+)/sugar cotransport. While the key characteristics of secondary active sugar transport were maintained in both modes, namely, Na(+) and voltage dependence, the kinetic properties of the two sides indicated a functional asymmetry of the transporter. That is, the apparent affinity for sugar and driver cation Na(+) exhibited a difference of more than 1 order of magnitude between the two modes. Furthermore, the selectivity pattern of ligands and the interaction of the transporter with the competitive inhibitor phlorizin were different. Whereas the high-affinity substrates, D-glucose and D-galactose, inhibited uptake of radioactive sugar tracer at their physiological concentrations (10 mM) in the forward reaction, they were poor inhibitors even at high concentrations in the reverse transport mode. Taken together, these results confirm the successful employment of E. coli to express and characterize a human membrane protein (hSGLT1), elucidating the functional asymmetry of this cotransporter.     

Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.     

Effects on Conformational States of the Rabbit Sodium/Glucose Cotransporter through Modulation of Polarity and Charge at Glutamine 457

The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na⁺ and glucose. Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C after reaction with methanethiosulfonate derivatives, but Na⁺ and sugar binding remain intact. To explore the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 were studied using chemical modification and the two-electrode voltage-clamp technique. Compared to wild-type SGLT1, Q457C exhibits ∼20-fold reduction in phloridzin affinity and preferential occupancy of an inward-facing state. Alkylation of Q457C by [(2-trimethylammonium) ethyl] methanethiosulphonate bromide, (MTSET), reverses these changes while blocking transport. Analysis of pre-steady-state currents in the absence of sugar yields three decay constants for each of Q457C, Q457C-MTSET and Q457R. Comparison of Q457C-MTSET and Q457R with Q457C and wild-type, reveals that inhibition of transport is accompanied by a decrease in magnitude and voltage-independence of the slow decay constant at negative potentials. But fast and medium decays remain unchanged. Computer simulation of transient currents suggests that introduction of positive charge at position 457 leads to a predominant outward rather than inward-facing conformational state. Taken together, the results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier.     

Light-dependent K(+) channels in the mollusc Onchidium simple photoreceptors are opened by cGMP

Light-dependent K(+) channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268-272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K(+) channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K(+) and 200 mM Mg(2+) to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K(+) concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants tau(o1), tau(o2) and tau(c1), tau(c2), respectively, similar to those of the light-dependent channel. In both the channels, tau(o1) and tau(o2) in ms ranges were similar to each other, although tau(c2) over tens of millisecond ranges was different. tau(o1), tau(o2), and the mean open time tau(o) were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of tau(o2) or tau(o). In both, the reversal potentials using 200- and 450-mM K(+)-filled pipettes were close to the K(+) equilibrium potentials, suggesting that both the channels are primarily K(+) selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K(+) pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K(+) channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.     

Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes.

Intramembrane charge movement was recorded in guinea pig ventricular myocytes at 19-22 degrees C using the whole-cell patch clamp technique. From a holding potential of -110 mV, the dependence of intramembrane charge moved on test voltage (Q(V)) followed the sum of two Boltzmann components. One component had a transition voltage (V) of -48 mV and a total charge (Qmax) of congruent to 3 nC/microF. The other had a V of -18 mV and a Qmax of 11 nC/microF. Ba2+ currents through Ca channels began to activate at -45 mV and peaked at congruent to -15 mV. Na+ current peaked at -35 to -30 mV. Availability of charge (in pulses from -70 to +10 mV) depended on the voltage of conditioning depolarizations as two Boltzmann terms plus a constant. One term had a V of -88 mV and a Qmax of 2.5 nC/microF; the other had a V of -29 mV and a Qmax of 6.3 nC/microF. From the Q(V) dependence, the voltage dependence of the ionic currents, and the voltage dependence of the availability of charge, the low voltage term of Q(V) and availability was identified as Na gating charge, at a total of 3.5 nC/microF. The remainder, 11 nC/microF, was attributed to Ca channels. After pulses to -40 mV and above, the OFF charge movement had a slow exponentially decaying component. Its time constant had a bell-shaped dependence on OFF voltage peaking at 11 ms near -100 mV. Conditioning depolarizations above -40 mV increased the slow component exponentially with the conditioning duration (tau approximately equal to 480 ms). Its magnitude was reduced as the separation between conditioning and test pulses increased (tau approximately equal to 160 ms). The voltage distribution of the slow component of charge was measured after long (5 s) depolarizations. Its V was -100 mV, a shift of -80 mV from the value in normally polarized cells. This voltage was the same at which the time constant of the slow component peaked. Qmax and the steepness of the voltage distribution were unchanged by depolarization. This indicates that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature effects on the presteady-state and transport-associated currents of GABA cotransporter rGAT1

The effects of temperature on the gamma-aminobutyric acid (GABA) uptake and on the presteady-state and transport-associated currents of the GABA cotransporter, rat gamma-aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage-clamp. Increasing temperature from 15 to 30 degrees C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady-state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady-state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q(max)) remained constant; the tau versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (alpha) and inward (beta) rate constants as functions of temperature showed that they are affected differently, with a Q(10)=3.4 for alpha and Q(10)=1.5 for beta. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate-limiting diffusional component on this process.     

Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a "shift" in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)